Throughout this application various publications are referred to in brackets. Full citations for these references may be found at the end of the specification. The disclosures of these publications are hereby incorporated by reference in their entirety into the subject application to more fully describe the art to which the subject invention pertains.
The recent development of bioorthogonal click chemistry has led to an explosion of interest in selective covalent labeling of biomolecules in cells and living organisms [1, 2]. In these labeling reactions one of the two bioorthogonal functional groups is first incorporated into target biomolecules via genetic [3] or metabolic approaches [4, 5]. A biophysical probe, functionalized in a complementary fashion, is introduced in a second step, allowing detection or isolation of the target of interest. To minimize perturbations to the physiological state of the cells or organisms probed, an ideal ligation reaction must proceed in water at neutral pH and at temperatures between 25 to 37° C. without any cytotoxic effects. Further, the reactive partners participating in this transformation must be inert to the native functional groups present in the biological system [6, 7].
Few chemical reactions satisfy both the bioorthogonal and click requirements. Discovered by Sharpless-Fokin/Meldal in 2002, the Cu(I)-catalyzed azide alkyne cycloaddition (CuAAC) is the quintessential bioorthogonal click reaction for chemical biologists (FIG. 1A) [8, 9]. This transformation is accelerated by approximately seven orders of magnitude compared to the uncatalyzed version [10]. As a ligand-assisted process, the reaction is further accelerated by Cu(I)-stabilizing ligands (i.e. tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine, TBTA, (FIG. 1B, 1C) [11]. The fast reaction kinetics and exquisite selectivity of CuAAC has gained it widespread utilization in chemical biology and materials science [12, 13].
To date, however, the use of CuAAC in living systems has been hampered by the toxicity associated with the catalyst formulations (CuSO4 or CuBr+sodium ascorbate+TBTA) [7]. TBTA, the ligand utilized in the optimized CuAAC conditions to stabilize the Cu(I) oxidation state, has very poor water solubility, which mandates the use of high Cu loading (0.2-1 mM) to achieve reasonable reaction rates. Free Cu(I) ions that escape from the coordination sphere of TBTA promote the generation of reactive oxygen and nitrogen species and induce detrimental consequences to cellular metabolism [14]. For example, Escherichia coli that incorporated azidohomoalanine into their outer membrane protein OmpC survived the initial treatment with 100 μM CuBr for 16 h, but were no longer able to divide [15]. Similarly, greater than 90% of mammalian cells underwent apoptosis and cell lysis within 20 min when treated with 1 mM Cu(I) under optimized CuAAC conditions. Zebrafish embryos exhibited a similar sensitivity to Cu(I). When embryos were treated with 1 mM CuSO4, 1.5 mM sodium ascorbate, and 0.1 mM TBTA ligand, all the embryos were dead within 15 min [7]. As presently formulated, labeling of biomolecules via CuAAC is not feasible in living systems.
To improve upon the biocompatibility of the azide-alkyne cycloaddition, Bertozzi and coworkers have developed a copper-free [3+2] cycloaddition by employing ring strains as an alternative means for alkyne activation [16, 17]. Among the cycloalkynes examined, a difluorinated cyclooctyne, DIFO [18], and a biarylazacyclooctynone, BARAC [19], showed rapid kinetics in biomolecular labeling experiments. DIFO-fluorophore conjugates are particularly sensitive for imaging azide-tagged glycans within complex biological systems, including live cells [18], C. elegans [20] and zebrafish embryos [21], with very low background fluorescence. However, recent in vivo studies revealed that DIFO-based probes bind to mouse serum albumin non-specifically [22]. In addition, the construction of these cyclooctyne-based probes usually involves multistep linear synthesis, which can be a challenge [18, 23]. A major goal in this field is to identify a new copper catalyst formulation that can promote rapid azide-alkyne cycloaddition in living systems without cytotoxicity.
The present invention addresses this need by providing a ligand that renders the CuAAC biocompatible and extends the application of CuAAC to label biomolecules in living systems. Therefore, the ligand of the present invention allows for the in vivo imaging or profiling of biomolecules. Additionally, the ligand of the present invention allows for the diagnosis and treatment of diseases.